KEYWORDS: Biosensors, Proteins, Control systems, Real time imaging, Modulation, Signal attenuation, Fluorescence resonance energy transfer, Fluorescent proteins, Image processing, Oxidation
Reversible oxidation is emerging as an important regulatory mechanism in protein tyrosine phosphorylation. Generation of hydrogen peroxide (H2O2), upon growth factor stimulation, is hypothesized to inhibit activity of protein tyrosine phosphatases (PTPs). This ensures that protein tyrosine kinases can elevate the steady-state level of protein tyrosine phosphorylation, which then allows propagation of the tyrosine phosphorylation signal. However, the effects of H2O2 on the kinetics of tyrosine phosphorylation signaling remain poorly understood, especially in living cells. Therefore, we used a genetically encoded Src kinase-specific biosensor based on fluorescence resonance energy transfer (FRET) to image the kinetics of the Src-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermal growth factor (EGF). We examined the kinetics under increased and decreased H2O2 levels. Through a straightforward, quantitative analysis method which characterized the signaling kinetics, we demonstrated that H2O2 modulated the amplitude and duration of the signal by inhibiting PTPs' activity. Our evidence also suggested the effect of H2O2 on Src activation is mediated by H2O2-dependent inhibition of PTPs. Furthermore, we provide evidence showing global elevation of intracellular H2O2 level attenuates EGF-induced Src signaling.
The Stagger Extension Process (StEP), a recombination of DNA technique, has been used as a rapid molecular
mutagenesis strategy. In this study, for obtaining the fluorescence proteins with new properties, six fluorescence proteins
(EYFP, EGFP, ECFP, mCitrine, mCerulean and Venus) were used as the templates to recombine the mutation library by
the Stagger Extension Process (StEP) technique. Through screening this mutation library, we have obtained some useful
new FPs which are different fluorescent properties with ancestor. These protein will extend fluorescent proteins
application.
KEYWORDS: Luminescence, Tumors, In vivo imaging, Sensors, Proteins, Fluorescence resonance energy transfer, Content addressable memory, Signal detection, Confocal microscopy, Imaging systems
Matrix metalloproteinases (MMPs) are a kind of secretory proteinases. Degradation of the extracellular matrix (ECM) by MMPs enhances tumor invasion and metastasis. To monitor MMPs activity and assess the MMP inhibitor effects in vivo, we constructed a plasmid that encoded a secretory fluorescent sensor named DMC (DsRed2-MSS-CFP expressed from pDisplay vector) that DsRed2 and cyan fluorescent protein (CFP) linked by MMP substrate site (MSS). MDA-MB 435s cells highly expressing endogenetic secretory MMP were transfected with the DMC plasmid so that the DMC could be cleaved by endogenetic MMP and the fluorescence ratio of DsRed2 to CFP was decreased. Treating the cells with GM6001, an MMP inhibitor, blocked the cleavage of DMC and caused an increase of the DsRed2/CFP ratio. The same result was achieved by using an in vivo tumor model that stable DMC-expressing MDA-MB 435s cells inoculated onto the chorioallantoic membrane of developing chick embryos to form primary tumors on the membrane. Thus, the fluorescent sensor DMC is able to sensitively monitor MMP activity and assess MMP inhibitors for anticancer research in vivo. This proves a novel method to efficiently screen and assess the anticancer drug MMP inhibitor in living cells and in vivo tumor models.
KEYWORDS: Luminescence, Tumors, Content addressable memory, Sensors, Fluorescence resonance energy transfer, Proteins, Signal detection, In vivo imaging, Optical filters, Cancer
Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) enhances tumor invasion and metastasis. To monitor MMP activity, we constructed plasmid that encoded a fluorescent sensor DC, in which an MMP substrate site (MSS) is sandwiched between DsRed2 and ECFP. MMPs are secretory proteins, only acting on the outside of cells; hence, an expressing vector was used that displayed the fluorescent sensor on the cellular surface. The DC was expressed in cells with high secretory MMP, so MSS was cleaved by MMP. Also, GM6001, an MMP inhibitor, causes DsRed2 signals to increase in living cells and on the chick embryo chorioallantoic membrane (CAM). Thus, this fluorescent sensor was able to sensitively monitor MMP activation in vivo. Potential applications for this sensor include high-throughput screening for MMP inhibitors for anti-cancer research, and detailed analysis of the effects of MMP
inhibitors.
Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also
involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes
simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its
effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and
TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance
energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an
enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with
efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and
the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was
due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced
apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.
Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.
Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also
involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its
effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and
TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance
energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an
enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with
efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and
the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was
due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the ACV-induced
apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.
Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.
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